Fig 2: Dyn2-GFP localizes to the bulb and not the neck of caveolae. (A) SIM image of a Dyn2-GFP-Cav1-mCh cell transiently expressing ?N?EH EHD2-BFP. Yellow arrows highlight Dyn2-GFP localization to Cav1-mCh at the tip of membrane tubes. Insets show magnification of the indicated area. Scale bar, 2 or 0.4 µm for magnification. The experiment was repeated three times. (B) Quantification of the fluorescent area of ?N?EH EHD2-BFP–positive tubes or Cav1-mCh–positive spots that were overlapping with Dyn2 fluorescence. Data are shown as scatter dot plot, mean ± SEM from three cells. Significance was assessed using t test, ****P = 0.0001. (C) Apparent FRET efficiency between GFP and mCh fluorophores in GFP-EHD2-Cav1-mCh cells, Dyn2-GFP-Cav1-mCh cells, or Cav1-mCh transiently expressing Cavin1-GFP. ROIs of structures positive for only GFP (green) or GFP and Cav1 (orange) were used to calculate the apparent FRET efficiency. Significance was assessed using t test, ****P = 0.0001.

Fig 3: Characterization of GFP-EHD2-Cav1-mCh and Pac2-GFP-Cav1-mCh HeLa FlpIn cells and localization of Dyn2-GFP following EHD2 or Pac2 depletion. (A) Representative image from TIRF movie of a GFP-EHD2-Cav1-mCh cell. Red arrow depicts structures only positive for Cav1-mCh, green arrow highlights spots only positive for GFP-EHD2, and yellow arrows highlight structures positive for both GFP-EHD2 and Cav1-mCh. Immunoblot of GFP-EHD2-Cav1-mCh cells treated with concentrations of doxycycline as indicated. Doxycycline concentration used for further experiments were 1.0 ng/ml. (B) Representative image from TIRF movie of Pac2-GFP-Cav1-mCh cell. Red arrow depicts structures only positive for Cav1-mCh, green arrow highlights spots only positive for Pac2-GFP, and yellow arrows highlight structure positive for both Pac2-GFP and Cav1-mCh. Immunoblot of Pac2-GFP-Cav1-mCh cells treated with concentrations of doxycycline as indicated. Doxycycline concentration used for further experiments was 1.0 ng/ml. (C) Immunoblots of cells treated with siRNAs as indicated. GAPDH served as a loading control. (D) Representative image from TIRF movie of Dyn2-GFP-Cav1-mCh cells treated with siRNA directed against EHD2 or Pac2 as indicated. Yellow arrows highlight caveolae positive for Dyn2-GFP. (E) Quantification of Cav1-mCh track mean speed in GFP-EHD2-Cav1-mCh cells (green). Numbers were related to Cav1-mCh cells (white). Track mean ± SEM from at least seven cells per condition are shown. Significance was assessed using t test, ***P = 0.001. All scale bars, 10 µm. (F) FLIM-FRET of GFP-fusion proteins. Top images show fluorescence micrographs of cells expressing GFP-fusion proteins and Cav1-mCh as indicated. Bottom of the two panels show EGFP fluorescence lifetime. White arrows highlight structures where EGFP and mCherry colocalize. Scale bar, 2 µm. Source data are available for this figure: SourceData FS3.

Fig 4: Dyn2 and EHD2 stabilize caveolae via different but cooperative mechanisms. (A) Image panel showing GFP recovery after photobleaching in Dyn2GFP-Cav1-mCh cells, GFP-EHD2-Cav1-mCh cells, or Pac2-GFP-Cav1-mCh cells. Graph shows percentage GFP-intensity recovery curves after photobleaching of mCh and GFP colocalizing structures at the PM. Arrowheads depict caveolae prior to and after photobleaching as indicated in the figure. Scale bar, 2 µm. (B) Recovery curves of Dyn2-GFP intensities after photobleaching of caveolae in Dyn2-GFP-Cav1-mCh cells treated with Ctrl, EHD2, or Pac2 siRNA as indicated. (C) Quantification of Cav1-mCh track duration time and displacement length in GFP-EHD2-Cav1-mCh cells (green). Numbers were related to Cav1-mCh cells (white). Track mean ± SEM from at least seven cells per condition are shown. (D) Representative image from TIRF movie of Dyn2-GFP-Cav1-mCh cells transiently expressing EHD2-BFP. White arrows highlight structures where all three tagged proteins are colocalized, and green arrows highlights structures only positive for Dyn2-GFP. Scale bar, 10 or 2 µm for magnification. (E) Immunogold labeling of EHD2 (large gold) and Dyn2 (small gold) on PM lawns prepared from 3T3-L1 cells. Arrowheads depict caveolae positive for both EHD2 and Dyn2. Scale bar, 100 nm. (F) Quantification of Cav1-mCh track duration time and displacement length in Dyn2-GFP-Cav1-mCh cells (black), transiently expressing EHD2-BFP (dark green), or depleted from EHD2 (purple). Numbers were related to Dyn2-GFP-Cav1-mCh cells (black). Track mean ± SEM from at least nine cells per condition are shown. Significance was assessed using t test, **P = 0.01, ***P = 0.001.

Fig 5: The stabilization of caveolae by Dyngo 4a is independent of Dyn2. (A) Color-coded trajectories of caveolae in Dyn2-GFP-Cav1-mCh cells pretreated with DMSO (Ctrl), Dyngo 4a, or Ryngo 1-23 for 30 min before imaged on TIRF over 5 min. Scale bar, 10 µm. (B) Quantification of percentage of caveolae positive for Dyn2-GFP after 30 min treatment with DMSO (black) or Dyngo 4A (gray). Data from at least 16 cells per condition are shown as scatter dot plot, mean ± SEM. (C) Quantification of Cav1-mCh track duration time and displacement length in cells as indicated. Numbers were related to ctrl-treated cells. Analysis was performed using Imaris software, and track mean from at least eight cells per condition are shown ± SEM. (D) Quantification of Cav1-mCh track duration time in Cav1-mCh cells treated with siRNA as indicated prior to pretreatment of DMSO (Ctrl) or Dyngo 4a as in A. Numbers were related to DMSO-treated ctrl siRNA-treated cells (black). Analysis was performed using Imaris software, and track duration time from at least eight cells per condition is shown as ± SEM. Significance was assessed using t test, ***P = 0.001.